Toan Pham

Mitochondrial inefficiencies and anoxic ATP hydrolysis capacities in diabetic rat heart

As ~80% of diabetic patients die from heart failure, an understanding of diabetic cardiomyopathy is crucial. Mitochondria occupy 35-40% of the mammalian cardiomyocyte volume and supply 95% of the hearts ATP, and diabetic heart mitochondria show impaired structure, arrangement, and function. We predict that bioenergetic inefficiencies are present in diabetic heart mitochondria; therefore, we explored mitochondrial proton and electron handling by linking oxygen flux to steady-state ATP synthesis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (ΔΨ) within rat heart tissues. Sprague-Dawley rats were injected with streptozotocin (STZ, 55 mg/kg) to induce type 1 diabetes or an equivalent volume of saline (control, n = 12) and fed standard rat chow for 8 wk. By coupling high-resolution respirometers with purpose-built fluorometers, we followed Magnesium Green (ATP synthesis), Amplex UltraRed (ROS production), and safranin-O (ΔΨ). Relative to control rats, the mass-specific respiration of STZ-diabetic hearts was depressed in oxidative phosphorylation (OXPHOS) states. Steady-state ATP synthesis capacity was almost one-third lower in STZ-diabetic heart, which, relative to oxygen flux, equates to an estimated 12% depression in OXPHOS efficiency. However, with anoxic transition, STZ-diabetic and control heart tissues showed similar ATP hydrolysis capacities through reversal of the F1F0-ATP synthase. STZ-diabetic cardiac mitochondria also produced more net ROS relative to oxygen flux (ROS/O) in OXPHOS. While ΔΨ did not differ between groups, the time to develop ΔΨ with the onset of OXPHOS was protracted in STZ-diabetic mitochondria. ROS/O is higher in lifelike OXPHOS states, and potential delays in the time to develop ΔΨ may delay ATP synthesis with interbeat fluctuations in ADP concentrations. Whereas diabetic cardiac mitochondria produce less ATP in normoxia, they consume as much ATP in anoxic infarct-like states.

Uncoupling of oxidative phosphorylation and ATP synthase reversal within the hyperthermic heart

Heart failure is a common cause of death with hyperthermia, and the exact cause of hyperthermic heart failure appears elusive. We hypothesize that the energy supply (ATP) of the heart may become impaired due to increased inner‐mitochondrial membrane permeability and inefficient oxidative phosphorylation (OXPHOS). Therefore, we assessed isolated working heart and mitochondrial function. Ex vivo working rat hearts were perfused between 37 and 43.5°C and showed break points in all functional parameters at ~40.5°C. Mitochondrial high‐resolution respirometry coupled to fluorometry was employed to determine the effects of hyperthermia on OXPHOS and mitochondrial membrane potential (ΔΨ) in vitro using a comprehensive metabolic substrate complement with isolated mitochondria. Relative to 37 and 40°C, 43°C elevated Leak O2 flux and depressed OXPHOS O2 flux and ∆Ψ. Measurement of steady‐state ATP production from mitochondria revealed decreased ATP synthesis capacity, and a negative steady‐state P:O ratio at 43°C. This approach offers a more powerful analysis of the effects of temperature on OXPHOS that cannot be measured using simple measures such as the traditional respiratory control ratio (RCR) or P:O ratio, which, respectively, can only approach 1 or 0 with inner‐membrane failure. At 40°C there was only a slight enhancement of the Leak O2 flux and this did not significantly affect ATP production rate. Therefore, during mild hyperthermia (40°C) there is no enhancement of ATP supply by mitochondria, to accompany increasing cardiac energy demands, while between this and critical hyperthermia (43°C), mitochondria become net consumers of ATP. This consumption may contribute to cardiac failure or permanent damage during severe hyperthermia.

Does the intercept of the heat–stress relation provide an accurate estimate of cardiac activation heat?

The heat of activation of cardiac muscle reflects the metabolic cost of restoring ionic homeostasis following a contraction. The accuracy of its measurement depends critically on the abolition of crossbridge cycling. We abolished crossbridge activity in isolated rat ventricular trabeculae by use of blebbistatin, an agent that selectively inhibits myosin II ATPase. We found cardiac activation heat to be muscle length independent and to account for 15–20% of total heat production at body temperature. We conclude that it can be accurately estimated at minimal muscle length.

Impaired ADP channeling to mitochondria and elevated reactive oxygen species in hypertensive hearts

Systemic hypertension initially promotes a compensatory cardiac hypertrophy, yet it progresses to heart failure (HF), and energetic deficits appear to be central to this failure. However, the transfer of energy between the mitochondria and the myofibrils is not often considered as part of the energetic equation. We compared hearts from old spontaneously hypertensive rats (SHRs) and normotensive Wistar controls. SHR hearts showed a 35% depression in mitochondrial function, yet produced at least double the amount of reactive oxygen species (ROS) in all respiration states in left ventricular (LV) homogenates. To test the connectivity between mitochondria and myofibrils, respiration was further tested in situ with LV permeabilized fibers by addition of multiple substrates and ATP, which requires hydrolysis to mediate oxidative phosphorylation. By trapping ADP using a pyruvate kinase enzyme system, we tested ADP channeling towards mitochondria, and this suppressed respiration and elevated ROS production more in the SHR fibers. The ADP-trapped state was also less relieved on creatine addition, likely reflecting the 30% depression in total CK activity in the SHR heart fibers. Confocal imaging identified a 34% longer distance between the centers of myofibril to mitochondria in the SHR hearts, which increases transverse metabolite diffusion distances (e.g., for ATP, ADP, and creatine phosphate). We propose that impaired connectivity between mitochondria and myofibrils may contribute to elevated ROS production. Impaired energy exchange could be the result of ultrastructural changes that occur with hypertrophy in this model of hypertension.

Pulmonary arterial hypertension reduces energy efficiency of right, but not left, rat ventricular trabeculae

Pulmonary arterial hypertension (PAH) triggers right ventricle (RV) hypertrophy and left ventricle (LV) atrophy, which progressively leads to heart failure. We designed experiments under conditions mimicking those encountered by the heart in vivo that allowed us to investigate whether consequent structural and functional remodelling of the ventricles affects their respective energy efficiencies. We found that peak work output was lower in RV trabeculae from PAH rats due to reduced extent and velocity of shortening. However, their suprabasal enthalpy was unaffected due to increased activation heat, resulting in reduced suprabasal efficiency. There was no effect of PAH on LV suprabasal efficiency. We conclude that the mechanism underlying the reduced energy efficiency of hypertrophied RV tissues is attributable to the increased energy cost of Ca2+ cycling, whereas atrophied LV tissues still maintain normal mechano‐energetic performance.

A flow-through infusion calorimeter for measuring muscle energetics during pharmacological interventions

We report on the design, development, and performance of a new calorimeter for measuring the heat-production and force-development of tiny samples of muscle tissue, while they are exposed to time-varying concentrations of a pharmacological agent. Fluid is delivered to a muscle sample by a syringe pump synchronized with a servo-controlled gravity-fed flow system. The rate of muscle heat production is inferred from voltages arising from a pair of thermopiles, to a resolution of approximately 6nW, providing a signal:noise ratio of approximately 1350. We demonstrate the utility of the instrument by selectively chemically disabling and forcibly engaging the muscles molecular force development mechanism, while measuring muscle heat-rate and force. These data reveal for the first time that the energetic cost of muscle activation is invariant with muscle length. In a separate experiment, we measure the total heat production of a muscle as it transitions into chemically-induced contracture.

Assessing the Efficiency of the Diabetic Heart at Subcellular, Tissue and Organ Level

In this review, we focus on the diabetic heart rather than the vascular complications of diabetes. Focus is further narrowed to a specific, but widely used, animal model: the diabetic rat heart in which diabetes has been induced by a single injection of streptozotocin. Our experimental approach is primarily biophysical and ranges from measurements made in isolated working whole-hearts, to those made from isolated left-ventricular tissues and mitochondria. Our interest is on the effect of severe diabetes on cardiac energetics, in terms of efficiency of cardiac work performance, ATP synthesis and oxygen consumption. By designing experiments to test the energetic performance of the heart and its trabeculae across a wide range of protocols, we have revealed the dependence of efficiency on afterload. This has allowed us to clarify a long-standing uncertainty in the literature; whereas the diabetic heart is unable to work against high afterloads, it nevertheless retains normal peak efficiency. But a further anomaly has been revealed. Whereas there is no evidence that the diabetic myocardium loses peak mechanical efficiency, its mitochondria demonstrate a decreased P:O ratio - i.e., a decreased bioenergetic efficiency. This decrease is consistent with an increase in the rate of production of reactive oxygen species, together with elevated proton leakage across the inner mitochondrial membrane at near maximal phosphorylating respiration states.

Left-Ventricular Energetics in Pulmonary Arterial Hypertension-Induced Right-Ventricular Hypertrophic Failure

Pulmonary arterial hypertension (PAH) alters the geometries of both ventricles of the heart. While the right ventricle (RV) hypertrophies, the left ventricle (LV) atrophies. Multiple lines of clinical and experimental evidence lead us to hypothesize that the impaired stroke volume and systolic pressure of the LV are a direct consequence of the effect of pressure overload in the RV, and that atrophy in the LV plays only a minor role. In this study, we tested this hypothesis by examining the mechanoenergetic response of the atrophied LV to RV hypertrophy in rats treated with monocrotaline. Experiments were performed across multiple-scales: the whole-heart in vivo and ex vivo, and its trabeculae in vitro. Under the in vivo state where the RV was pressure-overloaded, we measured reduced systemic blood pressure and LV ventricular pressure. In contrast, under both ex vivo and in vitro conditions, where the effect of RV pressure overload was circumvented, we found that LV was capable of developing normal systolic pressure and stress. Nevertheless, LV atrophy played a minor role in that LV stroke volume remained lower, thereby contributing to lower LV mechanical work output. Concomitantly lower oxygen consumption and change of enthalpy were observed, and hence LV energy efficiency was unchanged. Our internally consistent findings between working-heart and trabecula experiments explain the rapid improvement of LV systolic function observed in patients with chronic pulmonary hypertension following surgical relief of RV pressure overload.

Do right‐ventricular trabeculae gain energetic advantage from having a greater velocity of shortening?

We designed a study to test whether velocity of shortening in right‐ventricular tissue preparations is greater than that of the left side under conditions mimicking those encountered by the heart in vivo. Our experiments allowed us to explore whether greater velocity of shortening results in any energetic advantage. We found that velocity of shortening was higher in the rat right‐ventricular trabeculae. These results at the tissue level seem paradoxical to the velocity of ventricular ejection at the organ level, and are not always in accord with shortening of unloaded cells. Despite greater velocity of shortening in right‐ventricular trabeculae, they neither gained nor lost advantage with respect to both mechanical efficiency and the heat generated during shortening.